A The present invention relates to the field of immunoassays. The invention provides a simple and convenient method suitable for the measurement of a range of different types of analyte which occur, either totally or partially complexed with soluble binding proteins or receptors, in serum or plasma. The invention also relates to kits of reagents suitable for performing measurements of analytes using separation-based or homogeneous immunoassays.
For more than thirty years, immunoassays have been the method of choice for measuring low analyte concentrations in complex biological fluids. The procedure is equally applicable to the measurement is of low molecular weight compounds such as drugs, steroids and the like, as well as large molecular weight compounds such as protein molecules. The technique combines sensitivity and specificity and have been used in basic biological research to investigate the physiological and possible pathological role of a wide range of potent biologically active substances, including cytokines, steroid hormones, cyclic nucleotides, prostaglandins, leukotrienes and growth factors. Assay designs have proliferated over the last thirty years, as have the different types of signal reagents and detection systems. Sophisticated instruments with associated computer hardware have been developed with the aim of increasing sample throughput. Further background information relating to immunoassay techniques can be found in xe2x80x98The Immunoassay Handbookxe2x80x99, (Wild, D. G. Ed, Stockton Press, New York, [1994]).
The earliest methods were those which involved a step of separating the bound analyte from the free, in order to be able to measure the amount of bound analyte. Various separation methods have been described, including charcoal absorption, ammonium sulphate precipitation. More recently, solid supports have been utilised for immunoassay procedures, including magnetic particles and the walls of microtitre well plates. A recent development has been the introduction of homogeneous radioimmunoassay technology, e.g. the technique of scintillation proximity assays (SPA) covered by U.S. Pat. No.4,568,649.
As an alternative to radioisotopically-based immunoassay methods, non-radioactive systems have been introduced. Today, enzymes are the most widely used tracers in such immunoassay systems as ELISAs and EIAs. When used in combination with colourimetric end-points, they provide highly sensitive, robust, precise, accurate and convenient immunoassays. A major breakthrough came with the introduction of ninety-six well microtitre plates. Inexpensive automatic colourimetric multiwell plate readers are available. A number of other non-isotopic labels have been described, of which luminescent and fluorescent labels are the most popular.
Despite the widespread use of immunoassays, there are still difficulties in the measurement of analytes derived from particular sample types, notably plasma and serum, in which the analyte may be totally or partially complexed with a soluble receptor or binding protein. Engelberts et al (The Lancet, 338, 515xe2x88x9d6, 1991) have compared the results of the measurement of TNFxcex1 in plasma samples from septic shock patients using a range of assay types and have noted a marked variation in the amount of TNF detected. In this context, it has been suggested that TNF binding proteins present in biological fluids prevent the biological activity of TNF and cause discrepancies between various assay measurements. The difficulty in measuring analytes in the form of their complexes with soluble receptors or binding proteins, may be due to the masking of antibody binding sites.
This problem has been addressed previously by the development of an xe2x80x9coligoclonalxe2x80x9d assay system (Medgenix), in which up to three non-neutralising monoclonal antibodies are used in a standard ELISA assay for the measurement of cytokines. In this assay format, monoclonal antibodies are selected to recognise epitopes which are recognisably different from the binding site of the receptor or binding protein. As a result the assay is claimed to suffer no interference from soluble receptors or inhibitors and is capable of measuring total cytokine.
U.S. Pat. No. 4,121,975 (Ullman and Lavine) describes a method for pre-treatment of serum samples prior to the determination by immunoassay of polyiodothyronine, particularly thyroxine. In this assay, alkaline salicylate is added to serum as an agent to release thyroxine from its binding protein. Alpha-cyclodextrin is also included in the serum sample to complex naturally occurring, endogenous interfering molecules such as free fatty acids, lipids, and drugs. Cyclodextrin was not added to serum to sequester the salicylate or to safeguard the antibody components of the immunoassay. Moreover, the method is described for use with only one analyte.
U.S. Pat. No. 4,798,804 (Khanna and Pearlman) describe a method for serum pre-treatment prior to digoxin immunoassay. The method comprises contacting the serum sample to be analysed with xcex2-cyclodextrin or xcex2-cyclodextrin polymer in an amount and under conditions sufficient to allow a substantial portion of digoxin in the sample to bind to the xcex2-cyclodextrin. The xcex2-cyclodextrin-digoxin complex is separated from the other components of the medium by filtration or centrifugation, and the digoxin from the sample is thus highly concentrated. The digoxin is released from the xcex2-cyclodextrin complex using cyclohexanol to provide a digoxin sample which may be analysed by a number of immunoassay techniques.
In view of the needs of the prior art, the present invention provides a method of assaying for an analyte which occurs at least partially bound as a complex with its soluble receptor or binding protein. The method includes the step of forming a fluid mixture by mixing a biological fluid sample containing the analyte to be determined with a detergent for dissociating the complex. The method also includes mixing the fluid mixture obtained from the forming step with reagents, including a specific binding partner of the analyte for binding to the analyte, and performing a specific binding assay for the analyte. The method further includes the step of mixing the fluid mixture obtained from the forming step with a sequestrant and a detergent, whereby the specific binding assay of the mixing step is performed in the presence of the sequestrant. The present invention also provides a kit adaptable for performing the method of the present invention.